Process for the purification of poliovirus from cell cultures

ABSTRACT

The disclosure provides methods for poliovirus purification from crude cell culture harvests using a detergent followed by a clarification step.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/EP2015/066638, filed Jul. 21, 2015,designating the United States of America and published in English asInternational Patent Publication WO 2016/012445 A2 on Jan. 28, 2016,which claims the benefit under Article 8 of the Patent CooperationTreaty to European Patent Application Serial Nos. 14178399.3 and14178392.8, both filed Jul. 24, 2014.

TECHNICAL FIELD

The application relates to the field of biotechnology and virusproduction. More particularly, it concerns improved methods for thepurification of poliovirus particles from a cell suspension.

BACKGROUND

Recent developments in the field of vaccine production have created theneed for large-scale manufacturing. Robust and high-yield processes areneeded to support the world with sufficient amounts of (recombinant)vaccines to combat infectious diseases such as Polio.

Polioviruses are members of the Enterovirus genus of the familyPicornaviridae. Polioviruses are small, non-enveloped viruses withcapsids enclosing a single-stranded, positive sense RNA genome. Thereare three types of polioviruses: types 1, 2 and 3. Infections ofsusceptible individuals by poliovirus can result in paralyticpoliomyelitis. Poliomyelitis is highly contagious. Two different poliovaccines have been developed over time, the inactivated poliovirusvaccine (IPV) of Salk and the live attenuated oral poliovirus vaccine(OPV) of Sabin. Both vaccines are safe and effective. Each has itsparticular advantages and disadvantages, and both have played animportant role in the control of poliomyelitis. For a review aboutpolioviruses and polio vaccines, see, e.g., Kew et al., 2005.

The culture and purification systems for producing bulk poliovirusmaterial that can be used in a vaccine, in particular, for IPV,contribute to a large extent to the relatively high costs.

Thus, there remains a need in the art for efficient culture andpurification systems for producing poliovirus for use in vaccines; inparticular, there remains a need for purification processes forpolioviruses with high yields.

In a typical poliovirus production process, cells are grown in specificmedium and poliovirus is subsequently placed in contact with the cellsto allow the virus to infect the cells and to propagate. Afterpropagation of the poliovirus in the cells, the virus or componentsthereof are harvested from the cell culture.

One preferred currently used IPV manufacturing process usesanchorage-dependent, monkey-derived VERO cells that are grown onmicrocarriers and cultured in serum-supplemented media.

The virus produced and released in the cell culture medium can beseparated from the cellular biomass by conventional methods, such asdepth filtration and centrifugation. In such a case, the filtration orcentrifugation is the harvesting step. The filtered harvest is typicallyultra-filtrated to concentrate the viral suspension and, subsequently,the poliovirus can be purified, e.g., using gel filtration and/or ionexchange chromatography. Methods for harvesting and purifying poliovirusor viral components, and production of vaccines therefrom, have beenused in the art for decades already, and thus are well known and havebeen amply described, for example, in Van Wezel et al., 1978; Montagnonet al., 1984; WO 2007/007344 and U.S. Pat. No. 4,525,349, allincorporated by reference herein. The resulting concentrated virussuspension can optionally be diluted, and for preparing IPV, thepoliovirus therein will be inactivated, for which conventional methodscan be used.

The productivities of the currently used poliovirus production processesare not sufficient to gear up IPV production volumes needed foreradicating Polio on a worldwide scale. Hence, there is a limitation inthe global production capacity. In addition, the currently usedproduction processes, due to their low productivity, have high unitoperation costs because of large facility footprint and correspondinglyhigh medium and buffer consumption, together with high (bioactive) wasteproduction. Apart from costs associated with the vaccine manufacturingprocess, also product batch control and release costs scale withproductivity, i.e., high productivity batches significantly drive downcosts per vaccine dose.

One way of improving the yields of poliovirus production is to improvethe upstream production process. Processes for production of poliovirusat high yields have been achieved by increasing the cell density of theproduction cultures (see, e.g., WO 2011/06823), which, however, may poseadditional challenges in downstream processing. No developments forimproving poliovirus purification processes have been describedhitherto, either for lower or high-density cultures.

Therefore, there is a need in the industry for improved downstreamprocesses to further increase the yields of purification processes forpoliovirus.

BRIEF SUMMARY

This disclosure relates to improved methods of purifying poliovirusparticles from a crude cell harvest, and is also suitable for harvestswith high cell density. In certain exemplary embodiments, the methods ofthis disclosure may, for instance, have overall productivity rangingbetween 15-25, 6-12 and 10-16 dose IPV/ml virus culture,post-formaldehyde inactivation for poliovirus types 1, 2 and 3,respectively. This is a substantially higher volumetric yield than themethods known hitherto. Indeed, Kreeftenberg et al. (2006) haveestimated the overall yields for conventional, VERO cell platform-based,IPV processes to be 0.64, 1.04 and 0.34 dose/ml virus culture, based ona 40:8:32 D-antigen Units/dose for poliovirus types 1-3, respectively,and assuming an overall D-antigen recovery after inactivation of 40%.The noted significant increase in productivity will translate to eminentreduction of facility footprint with consequent lowering ofmanufacturing costs, while providing maximum capacity needed to supplythe global demand for IPV doses.

Downstream processing of high cell density suspensions using knownprocesses would commonly require a multitude of steps. A firstfiltration step would consist of a course filtration to remove wholecells, succeeded with a series of smaller size membrane filters toremove residual cell debris and precipitate material. Subsequently,after a concentration step, two or more selective chromatography stepsare required to obtain a sufficiently purified poliovirus suspensionaccording to regulatory requirements (WHO/EP).

It has been found and disclosed herein that directly after propagatingpoliovirus in a cell culture, the obtained crude cell culture harvestcontaining poliovirus could be treated with a detergent, preferablyselected from the group of cationic detergent, anionic detergent,non-ionic detergent and zwitterionic detergent in order to improve therelease of poliovirus into the harvest suspension. The so-obtainedoverall purification yields were unprecedented. Indeed, in certainembodiments, the process of this disclosure reached yields of 6-25 IPVdose/ml cell suspension as opposed to conventional Vero cell-basedplatforms yields of 0.3-1 IPV doses/ml cell suspension, obtained usingprocesses as disclosed hitherto.

The disclosure provides a method of purifying poliovirus from a crudecell culture harvest, the method comprising the steps of: a) adding adetergent to the crude cell culture harvest; and b) clarifying thepoliovirus-containing cell culture harvest to obtain a clarified harvestwith poliovirus particles.

The disclosure also provides a method of enhancing poliovirus releasefrom a crude cell culture harvest, the method comprising the steps of:a) adding a detergent to the crude cell culture harvest; and b)clarifying the poliovirus-containing cell culture harvest to obtain aclarified harvest with poliovirus particles.

The clarification step results in a clarified harvest, which comprises acontent strongly reduced in host cell DNA and cell debris, as comparedto the crude cell culture harvest.

Surprisingly, post clarification, a highly selective cationic exchangecapture step followed by a size separation-based polish step, i.e., sizeexclusion chromatography or diafiltration process step, was able toaccommodate for removal of high levels of Host Cell Protein (HCP)impurities from clarified harvests.

Therefore, this disclosure also provides a method of purifyingpoliovirus from a cell culture, the method comprising the steps of: a)adding a detergent to the cell culture; b) clarifying thepoliovirus-containing cell culture to obtain a clarified harvest withpoliovirus particles; and c) subjecting the clarified harvest obtainedin step b) to a capture step to obtain a poliovirus-containingsuspension. Preferably, the capture step is a cationic exchangechromatography step.

In a preferred embodiment, the poliovirus obtained in step c) of theprevious methods is further separated from the poliovirus-containingsuspension by size exclusion. Preferably, size exclusion is performed bysize-exclusion chromatography.

In a preferred embodiment, the disclosure also provides a method ofpurifying poliovirus from a cell culture, the method comprising thesteps of: a) adding a detergent to the cell culture; b) clarifying thepoliovirus-containing cell culture to obtain a clarified harvest withpoliovirus particles; c) subjecting the clarified harvest obtained instep b) to a cationic exchange chromatography step to obtain apoliovirus-containing suspension; and d) further purifying thepoliovirus from the poliovirus-containing suspension by size-exclusionchromatography.

The detergent used in this disclosure is preferably selected from thegroup of cationic detergents, anionic detergents, non-ionic detergentsand zwitterionic detergents. In an even more preferred embodiment, thedetergent is a cationic detergent, preferably the cationic detergent isselected from the group of Hexadecyltrimethylammonium bromide (CTAB),Hexadecylpyridinium chloride (CPC), Benzethonium chloride (BTC) anddomiphen bromide (DB). In a more preferred embodiment, the detergent isdomiphen bromide (DB).

In yet another embodiment, the preferred detergent is an anionicdetergent.

Preferably, the anionic detergent is selected from the group of Sodiumtaurodeoxycholate hydrate (STH) and Sodium dodecyl sulfate (SDS).

In yet another embodiment, the preferred detergent is a non-ionicdetergent. Preferably, the non-ionic detergent is selected from thegroup of 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON®X-100) and Decyl-β-D-1-thiomaltopyranoside (DTP).

In another embodiment, the preferred detergent is a zwitterionicdetergent. Preferably, the zwitterionic detergent is selected from thegroup of 3-(N,N-Dimethylmyristylammonio) propanesulfonate (SB3-14),3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).

This disclosure also provides for the use of a detergent for enhancingthe release of poliovirus from a crude cell culture harvest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (Panels A, B and C). D-antigen release from poliovirus-containingcrude cell culture harvests as a result of the treatment with adetergent (Domiphen bromide). Several harvests with distinct celldensities, each containing a different polio strain (Mahoney, MEF-1 orSaukett), have been treated with a detergent and subsequentlycentrifuged. The D-antigen concentration in the supernatant is disclosedas a function of the detergent concentration.

FIG. 2 (Panels A, B and C). Host cell DNA precipitation inpoliovirus-containing crude cell culture harvests as a result of thetreatment with a detergent (Domiphen bromide). Several harvests withdistinct cell densities and each containing a different polio strain(Mahoney, MEF-1 or Saukett) have been treated with a detergent andsubsequently centrifuged. The host cell DNA concentration in thesupernatant is disclosed as a function of the detergent concentration.

FIG. 3. Ratio D-antigen concentration in supernatant/D-antigenconcentration in crude, before and after treatment with a detergent.Ratios were measured for several harvests with distinct cell densitiesand containing different polio strains (Mahoney, MEF-1 or Saukett).

FIG. 4. Poliovirus purification flow chart.

FIG. 5. Product characterization by SDS-PAGE of poliovirus strains(Panel A) Mahoney, (Panel B) MEF-1, (Panel C) Saukett. 1=Marker,2=System suitability control, 3=CEX eluate, 4=SEC eluate.

FIG. 6. (Panel A) D-antigen release from poliovirus-containing crudecell culture harvests as a result of the treatment with differentcationic detergents; CTAB, CPC and BTC, respectively.

FIG. 6. (Panel B) Host cell DNA precipitation in poliovirus-containingcrude cell culture harvests as a result of the treatment with differentcationic detergents; CTAB, CPC and BTC, respectively.

FIG. 7 (Panels A, B and C). D-antigen release from poliovirus-containingcrude cell culture harvests as a result of the treatment with differenttypes of detergents (Anionic, zwitterionic and non-ionic detergents).

FIG. 8 (Panels A, B and C). Host cell DNA precipitation inpoliovirus-containing crude cell culture harvests as a result of thetreatment with different types of detergents (Anionic, zwitterionic andnon-ionic detergents).

DETAILED DESCRIPTION

This disclosure relates to improved methods of purifying poliovirusparticles from a crude cell culture harvest-containing poliovirus. Acrude cell culture harvest, as defined in this disclosure, is obtainedimmediately after cell culturing. It is referred to as “crude” becauseit has not been treated and has not been clarified in whatever formbefore being treated with a detergent. As opposed to the supernatant ofa cell culture harvest, the crude cell culture harvest contains cellsand cell debris, together with poliovirus particles.

In previously disclosed processes for poliovirus purification, e.g., inHenderson et al., a clarified harvest is treated as opposed to a crudecell culture harvest as described in the present application, thedifference being that the harvest in Henderson et al. already wentthrough a clarification step wherein the harvest was centrifuged toremove cell debris. In Henderson, a cationic detergent is not added to acrude cell culture harvest; instead, it is added to a clarified harvestfrom which cell debris has been previously removed.

In certain embodiments of this disclosure, the poliovirus particles arepurified from high cell density crude cell harvests, leading to highyields of purified poliovirus. These high cell density, crude cellculture harvests are obtained by culturing cells to high cell densities.Such culturing can, for instance, be performed in batch, fed-batch orperfusion mode. Methods for culturing cells to high cell densities areknown to the person skilled in the art. Specific methods for obtaininghigh cell density cultures are disclosed in, e.g., WO 2004/099396, WO2005/095578, and WO 2008/006494.

According to this disclosure, a high cell density, crude cell cultureharvest contains between about 5×10⁶ and 150×10⁶ cells/mL, e.g., betweenabout 8×10⁶ and 120×10⁶ cells/mL, e.g., between about 12×10⁶ and 100×10⁶cells/mL, e.g., between about 20×10⁶ and 80×10⁶ cells/m, e.g., betweenabout 10×10⁶ and 60×10⁶ cells/mL.

In a preferred embodiment of this disclosure, the cell density in thecrude cell culture harvest ranges between about 10×10⁶ and 50×10⁶cells/mL, e.g., at least about 15×10⁶ cells/mL, e.g., at least about20×10⁶ cells/mL, e.g., at least about 25×10⁶, e.g., up to about 30×10⁶cells/mL, e.g., up to about 35×10⁶ cells/mL, e.g., up to about 40×10⁶cells/mL, e.g., up to about 45×10⁶ cells/mL.

However, the methods according to this disclosure also work for harvestsfrom cell cultures with lower cell densities, e.g., between about0.5×10⁶ and 10×10⁶ cells/mL, e.g., between about 1×10⁶ and 5×10⁶cells/mL.

Typically, cell cultures are infected with poliovirus particles in orderto allow the poliovirus to propagate in the cells. Herewith, crude cellculture harvests are obtained that contain high concentrations ofpoliovirus in a single bioreactor. Methods for infecting cell culturesare known to the person skilled in the art. Specific methods forobtaining high cell density cultures with high virus concentration aredisclosed in, e.g., WO 2011/006823. This reference describes processesfor the production of large quantities of recombinant poliovirus. Theseprocesses rely on the ability to infect cultures at high cell densitywith preservation of a high poliovirus productivity per cell. Herewith,it offers a method to obtain a high cell density crude cell cultureharvest with high poliovirus concentrations in a single bioreactor.Typical yields of current processes for recombinant wild-typepoliovirus, infected at a cell density of, e.g., 12.5 million/ml andharvested 22-24 hours post-infection are in the range from 5.0×10⁹ to3.2×10¹⁰ TCID50/ml.

Once polioviruses have propagated in the cell culture, killing most ofthe cells (lysis), the poliovirus particles are, according to thisdisclosure, purified from the crude cell culture harvest.

Currently described processes for poliovirus production rely entirely onautologous release of poliovirus from the cells into the culture medium,which is a very efficient process for polioviruses. Surprisingly, it wasfound that a significant yield increase could be obtained when the cellculture (that already contained released poliovirus, cell debris, hostcell DNA and host cell proteins) was treated with a detergent,preferably selected from the group of cationic detergents, anionicdetergents, non-ionic detergents and zwitterionic detergents. Inaddition, this step resulted in a much cleaner clarified harvest withstrongly reduced host cell DNA and protein.

Assays Used to Quantify Host Cell DNA, Host Cell Proteins and PoliovirusParticles During the Process

Residual host cell DNA can be determined by real-time quantitative PCR,using quantitative Real-Time PCR with TAQMAN® probe. Primers and probeare designed for ribosomal 18S DNA. Quantities of sample DNA aredetermined by comparison to a DNA standard curve of known quantity thatis prepared from producer cell DNA. The standard curve DNA stock isdigested with the restriction enzyme Apa I to mimic sheared andpartially degraded DNA.

DNA of samples is isolated by treatment with deoxycholic acid andProteinase K. Real-Time PCR reactions are carried out using a FastReal-Time PCR system (ABI Prism 7500). DNA quantities are derived fromduplicate measurements of samples.

The concentration of residual host cell proteins (HCPs) was determinedin a commercially available enzyme-linked immunosorbent assay (ELISA)kit (Cygnus Technologies, F530), specific for HCPs. The concentrationswere determined in reference to the standard curve samples included inthe kit. The range of the assay is 25-200 ng/ml.

Polio vaccines are based on live virus or inactivated virus. Theycontain the poliovirus D-antigen, which is the important protectiveantigen. Virus yields can be measured by standard virus titrationtechniques, while the determination of the D-antigen concentration forMahoney, MEF-1 and Saukett poliovirus strains as a measure of potencycan be performed by a D-antigen enzyme-linked immunosorbent assay(ELISA). The assay is based on the binding of the D-antigen toserotype-specific antibodies to which mixture peroxidase reagent isadded. Peroxidase activity is then quantified by optical density. TheD-antigen concentrations are determined in reference to internationalIPV standard from the European Directorate for the Quality of Medicines& HealthCare (EDQM), see Fuchs et al. The assay range is 40-160 DU/mlfor Mahoney, 8-32 DU/ml for MEF-1 and 32-128 DU/ml for Saukett.

Immunogenicity can, for instance, be determined by in vivo testing inanimals. Potency can be determined using the D-antigen ELISA and by apoliovirus neutralizing cell culture assay on sera from previouslyimmunized rats.

Increased Release of D-antigen and Selective Precipitation of Host CellDNA

It was found that the addition of a detergent to a poliovirus-containingcrude cell culture harvest resulted in a substantial increase of theD-antigen concentration into the liquid phase of the harvest. At thesame time, it causes host cell DNA to precipitate. As exemplifiedherein, this precipitation step resulted in an increase of about 100% inD-antigen release from the crude cell harvest into the liquid phase andresulted in a reduction in host cell DNA of about at least 5 log 10following clarification.

Hence, this disclosure provides a method suited for purifying poliovirusparticles from a crude cell culture harvest and is also suitable for aharvest from a culture with a high cell density. The detergents that maybe useful in practicing this disclosure include, but are not limited to,cationic detergent, anionic detergents, non-ionic detergents andzwitterionic detergents.

In a preferred embodiment, the detergents that may be useful inpracticing this disclosure are cationic detergents, which include, butare not limited to, amine copolymers, quaternary ammonium compounds suchas, e.g., domiphen bromide (DB), Hexadecyltrimethylammonium bromide(CTAB), Hexadecylpyridinium chloride (CPC) and Benzethonium chloride(BTC), and any respective mixtures thereof. More specifically, the manyforms of polyethylenimine (PEI) have shown to be very effective inneutralization of excess anionic charge (DNA impurities). Appropriatecationic detergents for use in this disclosure include, but are notlimited to, the following classes and examples of commercially availableproducts: monoalkyltrimethyl ammonium salts (examples of commerciallyavailable products include cetyltrimethylammonium chloride or bromide asCTAB, tetradecyltrimethylammonium bromide or chloride (TTA),alkyltrimethyl ammonium chloride, alkylaryltrimethyl ammonium chloride,dodecyltrimethylammonium bromide or chloride,dodecyldimethyl-2-phenoxyethylammonium bromide, hexadecylamine chlorideor bromide salt, dodecyl amine or chloride salt, and cetyldimethylethylammonium bromide or chloride), monoalkyldimethylbenzyl ammonium salts(examples include alkyldimethylbenzyl ammonium chlorides andbenzethonium chloride as BTC), dialkyldimethyl ammonium salts(commercial products include domiphen bromide (DB), didecyldimethylammonium halides, and octyldodecyldimethyl ammonium chloride orbromide), heteroaromatic ammonium salts (commercial products includecetylpyridium halides (CPC or bromide salt and hexadecylpyridiniumbromide or chloride), cis-isomer1-[3-chloroallyl]-3,5,7-triaza-1-azoniaadamantane, alkyl soquinoliniumbromide, and alkyl dimethylnaphthyl-methyl ammonium chloride (BTC 1110),polysubstituted quaternary ammonium salts, (commercially availableproducts include, but are not limited to alkyldimethylbenzyl ammoniumsaccharinate and alkyldimethylethylbenzyl ammonium cyclohexylsulfamate),bis-quaternary ammonium salts (product examples include1,10-bis(2-methyl-4-aminoquinolinium chloride)-decane, 1,6-bis[1-methyl-3-(2,2,6-trimethyl cyclohexyl)-propyldimethyl ammoniumchloride] hexane or triclobisonium chloride, and the bis-quat referredto as CDQ by Buckman Brochures), and polymeric quaternary ammonium salts(includes polyionenes such aspoly[oxyethylene(dimethyliminio)ethylene(dimethyliminio)-ethylenedichloride],poly[N-3-dimethylammonio)propyl]N-[3-ethyleneoxyethylenedimethylammonio)propyl]ureadichloride, and alpha-4-[1-tri s(2-hydroxyethyl)ammonium chloride).

The skilled person will understand that these are examples of cationicdetergents, and based on the disclosure described herein, it is clearthat these will also be suitable in this application.

In an even more preferred embodiment, dialkyldimethylammonium salts suchas domiphen bromide (DB) are used in this disclosure. Though a largenumber of potential cationic detergents can be used to practice thisdisclosure, domiphen bromide is of particular interest due primarily toits availability as a GMP grade raw material and current use in otherproducts intended for human use. More specifically, since domiphenbromide is extensively used as an active ingredient in oral hygieneproducts as well as topical antibiotic creams, this molecule is producedin large quantities and released under cGMP conditions.

In another preferred embodiment, the detergents that may be useful inpracticing this disclosure are anionic detergents, which include, butare not limited to, alkyl sulfonates such as Sodium taurodeoxycholatehydrate (STH), 1-Octanesulfonic acid sodium salt, Sodium1-decanesulfonate, Sodium 1-heptanesulfonate and Sodium hexanesulfonate;and alkyl sulphates such as Sodium dodecyl sulfate (SDS), Lithiumdodecyl sulfate and Sodium octyl sulphate; and any respective mixturesthereof.

In yet another preferred embodiment, the detergents that may be usefulin practicing this disclosure are zwitterionic detergents that include,but are not limited to, 3-(N,N-Dimethylmyristylammonio)propanesulfonate(SB3-14), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS),3-[(3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate(CHAPSO), 3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt(SB3-8),3-[N,N-Dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate,3-(N,N-Dimethyloctadecylammonio)propanesulfonate (SB3-18),Amidosulfobetaine-14;3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate (ASB-14)and N,N-Dimethyldodecylamine N-oxide (DDAO); and any respective mixturesthereof.

In another preferred embodiment, the detergents that may be useful inpracticing this disclosure are non-ionic detergents that include, butare not limited to, poly(oxyethylene) ethers such as4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON® X-100),Polyethyleneglycol hexadecylether (BRIJ® 58), Polyethyleneglycolsorbitan monooleate (TWEEN®80),(1,1,3,3-Tetramethylbutyl)phenyl-polyethyleneglycol (TRITON® X-114),Polyoxyethylenesorbitan monolaurate (TWEEN®20), Polyethylene glycoldodecyl ether (Thesit); Glycosidic detergents such asDecyl-β-D-1-thiomaltopyranoside (DTP), 6-Cyclohexylhexyl β-D-maltoside(Cymal-6), Decyl-β-D-1-thioglucopyranoside, n-Dodecyl β-D-maltoside(DDM), Octyl β-D-glucopyranoside (OGP), Octyl β-D-1-thioglucopyranoside;Bile acids such as N,N-Bis[3-(D-gluconamido)propyl]deoxycholamide(Deoxy-BigCHAP); and any respective mixtures thereof.

The appropriate concentration of detergent for treating a polioviruscontaining high cell density suspension comprising a cell densityranging between 10×10⁶ and 150×10⁶ cells/mL ranges between about 1 mMand 12 mM. The appropriate concentration of DB for treating apoliovirus-containing high cell density suspension comprising a celldensity ranging between 10×10⁶ and 50×10⁶ cells/mL ranges between about1 mM and 4 mM. The appropriate concentration of DB for treating apoliovirus-containing high cell density suspension harvest comprising acell density ranging between 10×10⁶ and 30×10⁶ cells/mL ranges betweenabout 1 and 3 mM.

It will be within the purview of the skilled man in the art to testpotential substitutes for the detergents disclosed herein to identify acompound that effectively increases release of poliovirus from thecells, while it may further precipitate nucleic acid molecules and othercellular debris away from poliovirus particles as exemplified herein forHexadecyltrimethylammonium bromide (CTAB), Hexadecylpyridinium chloride(CPC), Benzethonium chloride (BTC) and domiphen bromide (DB) and at thesame time effectively precipitates nucleic acid molecules and othercellular debris away from poliovirus particles.

Therefore, this disclosure relates in part to methods of purifyingpoliovirus particles from a high cell density suspension whilesimultaneously enhancing virus recovery. The methods enhance the releaseof poliovirus particles and at the same time result in the selectiveprecipitation of host cell nucleic acid molecules away from thepoliovirus particles by adding a detergent to the crude cell cultureharvest.

Methods of Clarification

The crude cell culture harvest treated with a detergent is subsequentlyclarified to remove whole cells, precipitated host cell DNA, cell debrisand other impurities. Said clarification can be performed by depthfiltration. Centrifugation with or without polishing depth filtration isalso feasible. Therefore, the clarification of detergent-treated harvestmay be accomplished using centrifugation alone, or centrifugation intandem with a polishing clarification step such as depth filtration.

In choosing a filter or filter scheme, it is preferred to ensure arobust performance in the event upstream changes or variations occur.Maintaining the balance between good clarification performance and stepyields can be investigated by testing a variety of filter types withvarying internal media. Suitable filters may utilize cellulose filters,regenerated cellulose fibers, cellulose fibers combined with inorganicfilter aids (e.g., diatomaceous earth, perlite, fumed silica), cellulosefibers combined with inorganic aids and organic resins, or anycombination thereof, and polymeric filters (examples include, but arenot limited to, nylon, polypropylene, polyethersulfone) to achieveeffective removal and acceptable virus recoveries. In general, amultiple stage process is preferable but not required. An exemplary twoor three-stage process would consist of a coarse filter(s) to removelarge precipitate and cell debris followed by polishing second stagefilters(s) with nominal pore sizes greater than 0.2 μm but less than 1μm. The optimal combination will be a function of the precipitate sizedistribution as well as other variables. In addition, single stageoperations employing a relatively tight filter or centrifugation mayalso produce a product of good quality. More generally, anyclarification approach including dead-end filtration, microfiltration,centrifugation, or body feed of filter aids (e.g., diatomaceous earth)in combination with the dead-end or depth filtration, which provides afiltrate of suitable clarity to not foul the membrane and/or resin inthe subsequent step, will be acceptable to practice within thisdisclosure. Depth filtration shows a robust method of primaryclarification for this disclosure.

In another preferred embodiment according to this disclosure, an in-lineanionic exchange membrane can be integrated in the clarification filtertrain to further remove residual level of DNA or negatively chargedimpurities, like host cell proteins without loss of poliovirus underphysiological conditions. These high-capacity membranes with high pores(>3 um) are well known to effectively remove negatively chargedcontaminants like residual DNA, host cell proteins and/or adventitiousviruses in, for example, the monoclonal antibody production field.Suitable membranes are, among others, strong anionic exchange membraneswith positively charged ligand, typically quaternary ammonium grated to,for example, a cross-linked cellulose matrix.

Surprisingly, it was found that albeit typically viruses bind to thesemembranes under physiological conditions (pH 7-8, conductivity 15-18mS/cm), the poliovirus with a somewhat higher net isoelectric pointflows through these membranes, which makes it a suitable choice fornegatively charged impurity removal. In certain embodiments,chromatographic bead-based resins can be used but because of theadvantage of high convective flows, membranes are the preferred choice.

The combination of the detergent treatment and clarification stepsresults in a reduction in host cell DNA of at least 4 log 10, preferablyof at least 5 log 10, or even more preferably of at least 5.5 log 10.The overall pressure build up across the complete filter set up remainedbelow 0.8 bar, indicative for people skilled in the art that the filtertypes have been adequately selected and sized.

In a preferred embodiment, an anion exchange membrane can be integratedin the clarification filter train, eliminating the need for a dedicatedanionic exchange step, which is present in all currently knownpoliovirus manufacturing processes.

In preferred embodiments of the disclosure, harvested virus particlesare treated by the multi-stage clarification process flow that includes(in subsequent order): depth filtration via a charged filter unit (e.g.,a MILLIPORE® MILLISTAK® DOHC filter), dual membrane filter 0.8 μm/0.45μm (e.g., SARTOPORE® 2), a strong anionic exchange adsorption membrane(e.g., a SARTORIUS® Single Sep Q) and a sterile membrane filtration withrelatively small pore size (e.g., 0.22 μm filter units).

Steps for Further Purification

Following clarification, concentration of the clarified virus particlesuspension can be considered as a further step in the method accordingto this disclosure, but is by no means essential. Concentration of thevirus particle suspension can be performed by ultrafiltration. Thesuspension can be concentrated 5 to 20 times (and possibly be treatedwith nuclease, as mentioned hereinbelow). The particular ultrafiltrationmembrane selected will be of a size sufficiently small to retain virusparticles but large enough to effectively clear impurities. Depending onthe manufacturer and membrane type, nominal molecular weight cutoffsbetween 10 and 1000 kDa may be appropriate. The choice of molecular sizecutoff is dictated by the tradeoffs between yield and impurityclearance. Ultrafiltration using tangential flow mode is preferred. Inthis mode, the step may be controlled by setting a fixed cross-flow withor without backpressure on the retentate, setting a fixed transmembranepressure, or fixing both the cross-flow and the permeate flux.

Another process step that can be included at this stage of the process,but that is by no means essential, is the subsequent introduction of abuffer exchange process step via diafiltration. Diafiltration, or bufferexchange, using ultrafiltration units is used for removal and exchangeof salts, sugars and the like. The person skilled in the art knows underwhich conditions the buffer exchange should take place and which buffersare appropriate for this step.

Nuclease treatment can also be considered for inclusion in the processat this stage of the process, but is by no means essential. Nucleasetreatment can include the use of a broad spectrum of nucleases, e.g.,BENZONASE®, a DNase, an RNase, or any combination thereof. A nuclease orcocktail with both RNase and DNase activity is often used. A nucleasetreatment step can be contemplated at any point in the process, as longas residual nuclease content in the final product is acceptable to theapplication. The nuclease treatment may occur after clarification aloneor after clarification and a concentration step, but before a furtherpurification step such as a capture or polishing step.

An additional unit operation that can be used for impurity reductioncould, for example, be a chromatography unit, but is by no meansessential to the process. The chromatography unit consisting ofchromatography media in different formats such as resins, membraneadsorbers and monoliths can be used. The chromatography unitoperation(s) can be operated in either positive or negative mode(explained below). In certain embodiments of the disclosure, the virusparticle suspension fed to the chromatography unit(s) can be adjusted tocertain feed conditions such as, e.g., pH and/or ionic strength of thesolution. During this step, virus particles are further purified byseparating the virus particles from the remaining impurities such as,e.g., host cell nucleic acids and host cell proteins. Purification ofvirus particles during this step can be achieved by, e.g., affinitychromatography, anion exchange chromatography, cation exchangechromatography, size exclusion chromatography, reversed phasechromatography, hydrophobic chromatography, mixed mode chromatographyand/or hydroxyapatite chromatography, either used as a stand-aloneprocess step or in a combination with several process steps.

In certain embodiments of this disclosure, where chromatography unitoperation(s) are used, virus particles can be purified by separatingthem from the remaining impurities in the virus particle suspension.Virus particles can be either separated by binding the virus particlesat certain conditions to the chromatography media whereas some, if notmost of the impurities, are not bound to the chromatography media.Otherwise, virus particles can be separated by binding some, if not mostof the impurities to the chromatography media leaving most of the virusparticles unbound to the chromatography media. Above-mentioned operatingmodes are known in the art as positive binding mode and negative bindingmode, respectively.

It is also possible to operate certain chromatography unit operation(s)without any binding interaction depending on the chromatography mediaused. Exemplary chromatography media can be, but in no way limited to,size exclusion chromatography media (e.g., SEPHAROSE® 6FF). The personskilled in the art knows how to determine the required conditions forseparating virus particles from the impurities. Examples of thesequential use of different unit operations to achieve highly purifiedpolio virus solutions as above are described in literature, forinstance, in Bakker et al., 2011, and Thomassen et al., 2013.

In certain embodiments of the disclosure, chromatography unitoperation(s) can be used as the capture step, which is, in essence, acombination of a concentration and purification step, herewitheliminating the need for a stand-alone concentration step as describedpreviously (e.g., an ultrafiltration step). Chromatography media indifferent formats can be used in capture steps. Examples ofchromatography media formats are a.o. resins, membrane adsorbers andmonoliths. The person skilled in the art can easily determine theoptimal chromatography media format to be used for a particular processstep.

In certain embodiments of the disclosure, where chromatography unitoperation(s) are used as capture step(s), virus particles can bepurified by separating them from the remaining impurities in the virusparticle suspension. Virus particles can be separated by binding thevirus particles at certain conditions to the chromatography mediawhereas some, if not most, of the impurities are not bound to thechromatography media.

In certain embodiments of the disclosure, where chromatography unitoperation(s) are used as capture step(s), virus particle suspension,also referred to as the “feed material,” needs to be adjusted to certainconditions for optimal separation from the impurities. Exemplaryconditions of the feed material to be adjusted are, e.g., pH and ionicstrength of the virus particle suspension. The virus particles can befurther purified by the subsequent elution step that can be achieved by,e.g., changing the pH and/or ionic strength of the liquid phase of thechromatography medium.

In a particular embodiment of the disclosure, a cationic exchangechromatography medium is used as a capture step. The conditions of thefeed material can be adjusted by addition of acids or bases (e.g.,Citric Acid, NaOH) to reach desired pH and by addition of salt solutionsor de-ionized water (e.g., NaCl or MILLI-Q®) to reach desired ionicstrength. As a guide and certainly not a limitation, the pH couldpotentially range from about 4.5-7.0 and ionic strength can potentiallyrange from about 10 mS/cm-25 mS/cm. An extra clarification step (e.g.,0.45 μm or 0.22 μm filtration) after the feed material adjustment can beconsidered for inclusion in the process, in order to reduce the burdenon the following chromatography step, but is by no means essential. Theperson skilled in the art can easily determine what solutions andfiltration units to use for the particular process step.

In particular embodiments of the disclosure, where chromatography unitoperation(s) are used as capture step(s) and where virus particles areselectively bound to strong cationic exchange chromatography media forseparation, further virus particle purification is achieved by thesubsequent selective elution of the virus particles by changingconditions of the liquid phase within the unit operation. As a guide andcertainly not a limitation, elution of virus particles can be achievedby changing the pH of the liquid phase from acidic pH values to basic pHvalues (e.g., ranges between pH 4-10). As a guide and certainly not alimitation, elution of virus particles can also be achieved by changingthe ionic strength of the liquid phase from lower to higher ionicstrength (e.g., ranges between 10 mS/cm-35 mS/cm).

In a preferred embodiment of the disclosure, a poliovirus particlesuspension, also referred to as feed material, is adjusted to an acidicpH ranging from 4.4 to 5.6 and an ionic strength ranging from 14 mS/cmto 22 mS/cm. Subsequently, adjusted feed material is loaded to a cationexchange chromatography membrane adsorber (e.g., SARTOBIND® S) where thevirus selectively binds to the membrane. Virus particles are furtherpurified from impurities in the following elution step by increasing theionic strength of the elution buffer into the range between 25 mS/cm-40mS/cm while maintaining the pH range constant between 4.4-5.6. Elutedvirus particle suspension can subsequently be filtered through a, e.g.,0.22 μm filtration unit in order to reduce bioburden and precipitants.

According to this disclosure, if it is necessary for achieving a certainproduct purity, an additional purification step, called the “polishing”step, can be incorporated in the process. The “polishing” step is by nomeans essential in the whole purification process flow, but it is apreferred process step for achieving robustness in the wholepurification process flow.

During this step, it is desired to remove trace amounts of impuritiessuch as, e.g., but not limited to, host cell nucleic acids (e.g., DNA)and host cell proteins from the virus particle suspension. The“polishing” step can be achieved by, but is certainly not limited to,affinity chromatography, anion exchange chromatography, size exclusionchromatography, reversed phase chromatography, hydrophobicchromatography, mixed mode chromatography, hydroxyapatite chromatographyand/or ultrafiltration, either used as a stand-alone process step or ina combination of several process steps. During this step, bufferexchange of the virus suspension can be considered for inclusion to theprocess flow, but is by no means essential.

In certain embodiments of the disclosure, where chromatography unitoperation(s) are used as polishing step(s), chromatography media indifferent formats such as, e.g., resins, membrane adsorbers andmonoliths can be used. The chromatography unit operation(s) can beoperated in either positive or negative mode. In certain embodiments ofthe disclosure, the virus particle suspension fed to the polishingstep(s) can be adjusted to certain feed conditions such as, e.g., pHand/or ionic strength of the solution. The virus particles can bepurified by a subsequent elution step that can be achieved by, e.g.,changing the pH and/or ionic strength of the liquid phase of thechromatography medium.

In particular embodiments of the disclosure, virus particle purificationcan also be achieved by exploitation of size differences between thevirus particles and the impurities. Exemplary process steps can be sizeexclusion chromatography and/or ultrafiltration.

In particular embodiments of the disclosure, polishing step(s) can, inaddition to purifying the virus particles, be used as buffer exchangesteps. Exemplary process steps can be, but not limited to, sizeexclusion chromatography and/or diafiltration.

In particular embodiments of the disclosure, whereultrafiltration/diafiltration steps are incorporated, removal ofresidual impurities (e.g., host cell proteins, host cell nucleic acids)as well as exchanging the buffer to the desired buffer (e.g.,formulation buffer) can be achieved. Tangential flow ultrafiltration isuseful in removing residual protein and nucleic acid and to exchange thevirus particles into a formulation buffer. The selected ultrafiltrationmembrane will be of a size sufficiently small to retain virus particlesbut large enough to effectively clear impurities. Depending on themanufacturer and membrane type, nominal molecular weight cutoffs between100 and 1000 kDa may be appropriate.

In preferred embodiments of the disclosure, virus particles can beseparated from residual impurities by size exclusion chromatography(e.g., SEPHAROSE® 6FF) while concurrently, the buffer is exchanged to aformulation buffer. Desired levels of virus particle purity, as well asbuffer exchange quality, can be achieved by altering several variablesof the size exclusion chromatography unit. The person skilled in the artcan determine the optimal operating conditions in order to achieve therequired purity and process performance specifications.

A particularly preferred method to obtain purified poliovirus from cellculture according to the disclosure comprises the steps of: a) adding adetergent to the cell culture; b) clarifying the poliovirus-containingcell culture to obtain a clarified harvest with poliovirus particles; c)subjecting the clarified harvest obtained in step b) to a cationicexchange chromatography step to obtain a poliovirus-containingsuspension; and d) further purifying the poliovirus from thepoliovirus-containing suspension by size exclusion chromatography.

A sterile filtration step may be included at the end of the process inorder to reduce bioburden; such step is by no means essential. Theproduct can be filtered through a 0.22 μm modified polyvinylidenefluoride (PVDF) membrane (e.g., MILLIPORE® MILLIPAK®).

Scale of Cell Culture Systems and Downstream Processing Systems

The processes of this disclosure are scalable. The cell cultures forwhich this disclosure can be used range from small-scale cultures (e.g.,1-10 liter runs) to medium scale cultures (e.g., 20-1000 L runs) up tolarge commercial-scale preparations, such as 1000 to 50,000 L productionruns. The initial process steps such as depth filtration scale withculture volume while the cationic exchange chromatography or alternativecapture step and subsequent steps scale with poliovirus particle amount.Therefore, the batch size of the latter steps will be based on abioreactor productivity estimate of at least 5×10⁹ TCID50/m1 and up toabout 1×10¹¹ TCID50/ml. These high poliovirus yields can, for instance,be obtained by infecting high cell density cultures (as described, e.g.,in WO 2011/006823). The further purification of these high density cellsuspensions containing high concentrations of poliovirus particles ismade possible with this disclosure. The possibility to process thesesuspensions, which contain high amounts of cell debris and host cellimpurities allow for the purification of high quantities of poliovirusparticles per volume of suspension. It is the merit of this disclosureto provide for a method for processing cell culture batches with highcell densities, containing high concentrations of poliovirus particlesand therewith allowing for very high virus yields per processed volume.The present method, although it is applicable to large-scale cellcultures will allow for cells to be cultured at a smaller scale, yet tohigher cell densities and still reach high poliovirus yields that can beefficiently further processed. This method offers the possibility toprocess highly concentrated poliovirus batches that will have a greatimpact on the entire poliovirus purification industry.

Poliovirus and Producer Cells

A polio vaccine can be monovalent, containing one type of poliovirus(type 1, 2 or 3), or divalent (containing two types of poliovirus, e.g.,types 1 and 2, 1 and 3 or 2 and 3), or trivalent (containing three typesof poliovirus, i.e., types 1, 2 and 3).

It is possible to produce IPV from wild-type polioviruses.Alternatively, IPV may be produced from non-virulent live poliovirus,e.g., from the Sabin strains, which would further reduce the risk ofreintroducing wild-type poliovirus from IPV manufacturing (see, e.g., WO2007/007344, and Doi et al., 2001). This disclosure is suitable for thepurification of wild-type poliovirus (types 1, 2 and 3, e.g., the type 1strain Mahoney, type 2 strain MEF-1, or type 3 strain Saukett) as wellas of non-virulent types of poliovirus (e.g., the Sabin strains). Theprocesses according to the disclosure applied to produce IPV may serveto drive the cost down to such an extent that IPV may become availableto less- and least-developed countries. Although, in general, OPV ischeaper than IPV when prepared according to conventional methods, thehighly efficient processes of the disclosure can still reduce the costsof the bulk material for OPV and, hence, reduce the costs thereof aswell.

In general, each of the poliovirus strains is cultured in a separatebatch and if, for instance, a trivalent vaccine containing three typesof poliovirus is prepared, the (inactivated, for IPV) viruses are mixedand formulated for preparation of individual dosages. In certainembodiments, for example, a final vaccine per dose (e.g., 0.5 ml) may,for instance, comprise 40 D-antigen units (DU) of type 1 poliovirus, 8DU of type 2 poliovirus and 32 DU of type 3 poliovirus, determined bycomparison to reference preparations.

The method according to this disclosure can be applied to cell cultureharvest from distinct cell types. One type of cells that can be used inthe methods of this disclosure are PER.C6® cells, which are immortalizedcells, also known in the art as continuous cell lines, and as such, havethe potential for an infinite lifespan (see, e.g., Barrett et al.,2009). For the purpose of the present application, “PER.C6® cells” shallmean cells as deposited under ECACC no. 9602240 on 29 Feb. 1996. It willbe clear to the skilled person that this definition will include cellsfrom an upstream or downstream passage or a descendent of an upstream ordownstream passage of these deposited cells. PER.C6® cells are describedin U.S. Pat. No. 5,994,128 and in Fallaux et al., 1998. These cells arevery suitable for poliovirus production to produce cell-based poliovirusvaccines, since they can be infected and propagate the virus with highefficiency, such as, for instance, described in WO 2011/006823. It isdemonstrated herein that these cells are also very suitable forproduction of poliovirus to high levels in serum-free suspensioncultures.

Since other cell types can be used to propagate polioviruses, themethods of this disclosure are also applicable to processpoliovirus-containing crude cell harvests comprising other cell types.As exemplified herein, harvests from Vero cells and MRC-5 cells wereprocessed with the methods of this disclosure.

For large-scale manufacturing of inactivated polio vaccines, poliovirusis generally propagated on adherent Vero cells, which aremonkey-derived. Vero cells, which are cultured on microcarriers, arewidely used for vaccine production, including inactivated as well aslive attenuated polio vaccines, and thus far, are the most widelyaccepted continuous cell lines by regulatory authorities for themanufacture of viral vaccines, and use of these cells for vaccineproduction is expected to rise by experts in the field (Barrett et al.,2009). Large-scale microcarrier culture of Vero cells for inactivatedpoliovirus vaccine has been described by Montagnon et al., 1982 and1984. A process for the large-scale production of a polio vaccine usingVero cells, and the resulting vaccine, are also described in U.S. Pat.No. 4,525,349.

High titers of poliovirus (Sabin type 1) production (almost 2×10⁹TCID₅₀/ml) have been obtained in Vero cells cultured on microcarriers inserum-containing medium prior to the virus production phase, which tookplace in serum-free medium (Merten et al., 1997). In view of thedisadvantages of using serum, the authors have indicated that acompletely serum-free process is desired. Under serum-free conditions, apoliovirus production titer of 6.3×10⁸ TCID₅₀/ml was obtained. Thepoliovirus production titers obtained by the method of this disclosureon PER.C6® cells were ranging between 5.0×10⁹ to 3.2×10¹⁰ TCID50/ml (ata cell density at infection of 12.5 million/ml).

A conventional alternative cell platform commonly used for vaccineproduction, in general, and IPV production, in particular, are HumanFetal Lung Fibroblast Cells (MRC-5 cells) initiated by J. B. Jacobs,1966. A host cell line comparison study (Vlecken et al., 2013) showedthat adherent MRC-5 and VERO cell lines are the highest producers amongan extended host cell panel, which makes them suitable candidates forviral vaccine production. Using flask surface adherent cultures, virustiters achieved were (0.7-2.6)×10⁶ TCID50/ml and (1.4-5.8)×10⁶ TCID50/mlfor MRC-5 and Vero cell cultures, respectively.

The purification methods of the disclosure are suitable for polioviruspropagated in any cell type amenable for poliovirus propagation, i.e.,the methods of the disclosure are independent from the cell type usedfor growing poliovirus.

The disclosure is further explained in the following examples. Theexamples do not limit the disclosure in any way. They merely serve toclarify the disclosure.

EXAMPLES Example 1 Increased Poliovirus Purification Yields fromPoliovirus-containing Crude Cell Culture Harvest by Addition of aCationic Detergent

Cells from the PER.C6® cell line were grown in a serum-free culturemedium in a 10 L bioreactor operated in perfusion mode to a cell densityof approximately 50×10⁶ viable cells/ml (vc/ml). Prior to infection withpoliovirus type 1 (Mahoney), type 2 (MEF-1) or type 3 (Saukett), theculture was diluted with fresh culture medium to viable cell density inthe range between 12.5×10⁶ and 50×10⁶ vc/mL. The batch infection processtook place in 10 L bioreactors at 35° C. at a multiplicity of infectionof 1. At the time of harvest, 20-24 hours post-infection, a 50 ml samplewas taken, which was subsequently distributed in 11 aliquots of 4 mL.

In order to determine the effect of a detergent on thepoliovirus-containing cell culture harvests, a titration experiment wasperformed with Domiphen bromide (DB). A discrete amount of DB stocksolution was added to the sample aliquots at a targeted DB concentration(between 0 and 4 mM). The samples were mixed and incubated for one hourat 35° C. Subsequently, the samples were centrifuged for 5 minutes at3000 g to spin-down the precipitated DNA. Supernatant samples wereanalyzed for virus quantity by D-antigen ELISA, and for host cell DNAusing Q-PCR.

FIG. 1 (Panels A, B and C) shows D-antigen release frompoliovirus-containing cell culture harvests as a result of the treatmentwith a detergent (DB). Several harvests with distinct cell densities andeach containing a different polio strain (Mahoney, MEF-1 or Saukett)have been treated with a detergent and subsequently centrifuged. TheD-antigen concentration in the supernatant, which is corrected for thedetergent addition dilution, is given as a function of the detergentconcentration. FIG. 1 shows that after the addition of a detergent (DB),the virus titer increased substantially as compared to before theaddition of a detergent (DB). For each strain and for each viable celldensity, the same pattern can be observed, i.e., increasing thedetergent (DB) concentration leads to increased virus release from thecrude cell harvest into the liquid phase.

FIG. 2 (Panels A, B and C) shows host cell DNA precipitation inpoliovirus-containing crude cell culture harvests as a result of thetreatment with a detergent (Domiphen bromide). The concentrations on they-axis have been corrected for the detergent dilution factor. For eachstrain and for each viable cell density, host cell DNA is precipitatedfrom the crude cell culture harvest. FIG. 2 clearly indicates thateffective DNA clearance occurred in the aliquots for detergent (DB)concentrations above 1.3 mM.

Also, a minimum DB concentration could be determined from eachindividual curve (FIGS. 1 and 2), for which a plateau level of D-antigenis obtained and at the same time, maximum DNA clearance is obtained.This minimum amount of detergent increases with cell density toaccommodate for higher amounts of cells and increased level of solublehost cell DNA in the medium.

Since the increase of detergent did not lead to poliovirusprecipitation, a person skilled in the art would extrapolate theseresults to poliovirus-containing cell suspensions of even higher celldensities, e.g., of about 70×10⁶ cells/mL, e.g., of about 90×10⁶cells/mL, e.g., up to about 120×10⁶ cells/mL, e.g., up to about 150×10⁶cells/mL. The skilled person would conclude that the poliovirus fromsuch high cell density crude cell culture harvests can be purified bythe methods of this disclosure.

Example 2 Efficacy of Detergent Treatment on Poliovirus Release FromVERO and MRC-5 Crude Cell Culture Harvests

Treatment of a Crude Poliovirus Harvest from an Adherent VERO CellCulture

Vero cells were pre-cultured in T-175 flasks and scaled up to inoculate1 spinnerflasks Cytodex 3, at 30×10³ cells/cm² in VERO spinner medium(MEM+10% FBS+6 mM Glutamine+4.6 g/L Glucose) and 5 g/L microcarriersCytodex 3. The cells were incubated at 37° C., 5% CO₂ and stirred at 60rpm for the first 24 hours and at 90 rpm during the following days. Atday 3 post-seeding (on microcarriers), the cells were washed withpre-warmed PBS and a medium change was performed with infection medium(MEM+4 mM Glutamine). The replenished cell culture was distributed over50 ml tubespins containing 20 ml cell culture seeded at 1×10⁶ cells/mL.The tubespins were infected for each of the three virus strains(Mahoney, MEF-1, Saukett) with an MOI of 1. Infection was performed at35° C., 170 rpm, 5% CO₂ and the virus was harvested 72 hourspost-infection. In order to determine the effect of a detergent on thepoliovirus-containing crude VERO cell harvests grown on microcarriers, aDB stock solution was added to the harvest at a final concentration of1.6 mM. For this experiment, a DB stock solution (1.05% w/v, 40 mM NaCl)was used. The samples were mixed and incubated for one hour at 35° C.Subsequently, the samples were centrifuged for 5 minutes at 3000 g tospin down the precipitated DNA. Supernatant samples were analyzed forvirus quantity by D-antigen ELISA and for host cell DNA using Q-PCR.

Treatment of a Crude Poliovirus Harvest from an Adherent MRC-5 CellCulture

MRC-5 cells were cultured in BME+10% FBS (nHI)+4 mM Glutamine andincubated at 37° C. and 10% CO₂ in a T75 flask. Every 3 to 4 days, whencultures were approximately 80-90% confluent, MRC-5 cultures werepassaged and expanded into T-175 flasks. When the cells reached adensity of about 80-90% (day 4), the cells were washed with PBS and amedium change was performed with BME+4 mM Glutamine. T-175 flasks wereinfected for each of the three virus strains (Mahoney, MEF-1, Saukett)with an MOI of 1, in a total volume of 25 ml per flask. The infectionwas performed at 35° C., 10% CO2 and the virus was harvested 72 hourspost-infection. In order to determine the effect of a detergent on thepoliovirus-containing crude MRC-5-adherent cell harvests, a DB stocksolution was added to the harvest at a final concentration of 0.6 mM.The samples were mixed and incubated for one hour at 35° C.Subsequently, the samples were centrifuged for 5 minutes at 3000 g tospin down the precipitated DNA. Supernatant samples were analyzed forvirus quantity by D-antigen ELISA and for host cell DNA using Q-PCR.

TABLE 1 D-antigen concentration in supernatant of cell culture harvest[DU/ml] Vero-adherent MRC-5-adherent cell culture grown cell culturegrown on microcarriers on T-175 flasks DB concentration 0 mM 1.6 mM 0 mM0.6 mM Type 1 (Mahoney) 153 160 30 36 Type 2 (MEF-1) 19 34 8 13 Type 3(Saukett) 106 119 11 16

Table 1 shows the concentration of D-antigen upon DB treatment, at a DBconcentration of 1.6 mM for VERO cell poliovirus harvests and 0.6 mM forMRC-5 cell poliovirus harvests. The D-antigen concentrations in thetable are corrected for dilution caused by the detergent addition.

The results show that the addition of detergent (DB) caused additionalvirus to be released in the liquid phase of the VERO and MRC-5 cellculture harvests, whereas DNA is precipitated away from the virus.Indeed, DNA clearance at 1.6 mM DB was more than 2 log 10 in the VEROcell culture. DNA clearance at 0.6 mM DB was more than 3 log 10 in theMRC-5 cell culture (data not shown). This demonstrates that thedisclosure is applicable for various cell types used for poliovirusproduction.

Example 3 Impact of Detergent Treatment on Poliovirus Release and DNAClearance in a Bioreactor Prior to Cell Clarification

PER.C6® cells were grown in a serum-free culture medium in a 10 L glassbioreactor operated in perfusion mode to a cell density of approximately50×10⁶ vc/ml. Prior to infection with poliovirus type 1 (Mahoney), type2 (MEF-1) or type 3 (Saukett), the culture was diluted with freshculture medium to a viable cell density of 12.5×10⁶ vc/ml. The batchinfection process took place in 10 L bioreactors at 35° C. at amultiplicity of infection of 1. At the time of harvest, 20-24 hourspost-infection, a Domiphen Bromide (DB) stock solution was added in 30minutes while stirring to reach a final DB concentration of 2.2 mM DB.After detergent addition, the bioreactor was left to incubate for onehour at 35° C., while constantly stirring.

Samples of the crude cell harvest (without DB treatment and post-DBtreatment) were centrifuged (3000 g, 5 minutes) to sediment the cells.The crude samples (not centrifuged prior to DB treatment, thuscontaining the cells) and the supernatant samples were analyzed forpoliovirus and host cell DNA quantification, using a D-antigen ELISA andQ-PCR, respectively.

The results depicted in FIG. 3 show that in all runs (for all threestrains), the detergent treatment resulted in a two-fold increase ofD-antigen release, from the crude poliovirus harvest into the liquidphase. In addition, DNA was effectively precipitated by the treatmentwith DB. In all runs, the DNA clearance in respect to crude harvest wasmore than 5 log after detergent treatment, as opposed to 2 log prior todetergent treatment (data not shown). This demonstrates that thedisclosure can also be used at bioreactor scale.

The addition of a detergent had been used previously to remove host cellDNA in the field of adenovirus purification processes, as disclosed in,e.g., U.S. Pat. No. 7,326,555 and WO 2011/045378. However, selective DNAprecipitation has not been disclosed hitherto in the field of polioviruspurification. Polioviruses and adenoviruses are very distinct viruses.Indeed, a poliovirus is composed of a single-stranded RNA genomeencapsulated with a protein capsid and the viral particle is about 30nanometers in diameter. In contrast, adenoviruses represent the largestnon-enveloped viruses, with a diameter of about 90-100 nm. The proteincapsid of the adenovirus contains a double-stranded DNA helix and isuniquely populated with fibers or spikes that aid in attachment to thehost cell, which are absent in polioviruses. The isoelectric point ofadenoviruses is around pH 5.5, which means that the virus is negativelycharged under physiological conditions. A review article about theisoelectric point of poliovirus suggests that its value is higher thanfor adenoviruses pH 5.8-7.5 (Thomassen et al., 2013). As size and chargeare key determinants in chromatography and precipitation processes, itcould not have been predicted that the treatment with a detergent wouldhave a similar effect on a poliovirus-containing crude cell cultureharvest as it has on an adenovirus-containing crude cell cultureharvest.

More importantly, the unexpected effect of detergent treatment on therelease of poliovirus particles from the crude cell culture harvest intothe liquid phase of the harvest had not been observed in thepurification methods for adenoviruses. Thus, this surprising effectcould not have been foreseen based on previously used virus purificationmethods.

Example 4 Poliovirus Purification Process with and without DetergentTreatment and Impact on D-antigen Recovery and DNA Clearance

PER.C6® cells were grown in a serum-free culture medium in a 10 Lbioreactor operated in perfusion mode to a cell density of approximately50×10⁶ vc/ml. Prior to infection with poliovirus serotype 1 (Mahoney) ortype 3 (Saukett), the culture was diluted with fresh culture medium to aviable cell density of 11×10⁶ vc/ml and 9.5×10⁶ vc/ml, respectively. Thebatch infection process took place in 10 L bioreactors at 35° C. at amultiplicity of infection of 1. At the time of harvest, 22 hourspost-infection, two 1.5 L bulk samples were taken from the bioreactorand transferred into 2 L bottles. One bottle was taken to perform directfiltration; the other was treated with a detergent (DB) and subsequentlysubjected to filtration.

DB treatment was performed in a 2 L bottle at room temperature. DB stocksolution was added via a pipette in 30 equal portions in 30 minuteswhile stirring to reach a final DB concentration of 2.1 mM. Afterdetergent addition, the bottle was left to incubate for two hours whilemixing. Cell clarification was performed by passing the untreated crudeharvest or DB-treated harvest through a series of filters, i.e., apositively charged depth filter (MILLIPORE® MILLISTAK+® HC POD DOHC)with a pore size distribution of 4-8/0.6-1.5 μm, followed by twoconsecutive polyether sulfon (PES) membrane filters of reducing size0.8/0.45 μm (SARTORIUS®, SARTOPORE® 2) and 0.22 μm (MILLIPORE®,MILLIPAK®). During filtration, the first received filtrate wasdiscarded, then filtrate was collected in a product bottle until thefeed bottle was empty. Recovery of the virus was completed by additionof 1 system volume of PBS to the collected filtrate. The clarifiedharvest was analyzed for virus quantity, host cell DNA and HCP using aD-antigen ELISA, Q-PCR and host cell-specific protein ELISA,respectively. The impact of DB treatment on the performance of theharvest process is depicted in Table 2. Recovery is calculated withrespect to a whole broth sample taken from the crude harvest at the timeof harvest.

Consistent with the previous examples, the D-antigen recovery aftertreatment with a detergent (domiphen bromide) was significantlyincreased compared to the process without domiphen bromide. As a result,the volumetric productivity of the process was significantly increased.Indeed, the concentration of D-Antigen in the clarified harvest wasdoubled after detergent (DB) treatment.

Furthermore, clearance of HC-DNA by the detergent treatment step wasobserved, which was in accordance with the results described in previousexamples. According to Table 2, the treatment of a crude cell harvestcontaining poliovirus with a detergent (DB) helped to clear DNA by afactor of 1000. Moreover, it shows that Host Cell Proteins (HCP) werepartly removed by the use of detergent.

TABLE 2 Clarification of 1.5 L crude virus harvest with (+) and without(−) DB treatment for two virus strains Strain Mahoney Saukett Viablecell density at infection 11 9.5 DB treatment − + − + Clarified harvestD-antigen concentration (DU/ml) 1439 2977 510 1195 HC-DNA concentration(pg/ml) 1731 <0.4 702 <0.4 HCP concentration (μg/ml) 79 52 76 55D-antigen harvest recovery (%) 57 110 52 122 DNA log removal 1.9 >5.52.1 >5.3 HCP removal (%) 25 54 38 55

Example 5 DB Treatment as Part of Drug Substance ManufacturingInactivated Poliovirus Vaccine (IPV) Process

This example demonstrates the purification of wild-type poliovirusserotypes (Mahoney, MEF-1 and Saukett) from a crude cell culture harvestat 20 L scale. The downstream process steps involved are depicted inFIG. 3.

PER.C6® cells were grown in a serum-free culture medium in a 10 Lbioreactor operated in perfusion mode to a cell density of approximately50×10⁶ vc/ml. Prior to infection with poliovirus serotype 1 (Mahoney),type 2 (MEF-1) or type 3 (Saukett), the culture was divided over threebioreactors and diluted with fresh culture medium to a viable celldensity of 12×10⁶ vc/ml, 11×10⁶ vc/ml and 13×10⁶ vc/ml, respectively.The batch infection process took place in 10 L bioreactors at 35° C. ata multiplicity of infection of 1.

At the time of harvest, 23 hours post-infection, DB stock solution wasadded to the bioreactors over a period of 30 minutes, to a final DBconcentration of 2.2 mM DB. After detergent addition, the DB-treatedharvest was mixed for 60 minutes. Subsequently, clarification wasperformed by passing the DB-treated harvest through a series of filters,i.e., two 8-4/1.5-0.6 μm MILLISTAK® DOHC POD filters in parallel,followed by a 0.8/0.45 μm SARTOPORE® 2 filter, a Single Sep Q filterand, finally, a 0.22 μm MILLIPAK® filter.

The clarified harvest of two filtrations was pooled, acidified to pH 5.0and diluted to conductivity 11 mS/cm and filtered over a SARTORIUS®SARTOPORE® 0.8/0.45 μm filter prior to loading to a SARTORIUS®SARTOBIND® S membrane. The virus was retrieved from the membrane by stepelution using PBS. In the final step, the cation exchange (CEX) virusfraction was loaded on a column packed with SEPHAROSE® 6FF sizeexclusion chromatography resin with fractionation range 10-4000 kDa.During isocratic elution, residual HCPs were separated from the virusfraction pool, and also the matrix of the poliovirus was fully exchangedto a phosphate buffer containing NaCl.

Following purification, the purified virus solution was further dilutedwith SEC elution buffer to a preset absorbance unit (OD 260 nm), thenM199 and glycine (final concentration 5 g/L) were added and the fluidwas filtered over a 0.22 μm pore size filter prior to formaldehydeinactivation.

Inactivation was performed using 0.025% formalin during 13 days (with inbetween 0.22 μm filtration) at 37° C. according to the World HealthOrganisation (WHO) and European Pharmacopeia (EP) requirements.

In the above-described process, the main product intermediates, crudeharvest, clarified harvest, SEC eluate and inactivated polio virus (IPV)were analyzed for virus quantity, host cell DNA and total protein (TP)using a D-antigen ELISA, Q-PCR and Bradford assay, respectively.

Results and Discussion

Table 3 summarizes the quality attributes and yields of purifiedpoliovirus per serotype. Residual specific protein and DNA concentrationmeet regulatory requirements (WHO/EP). In addition, the absorbance ratioOD260/OD280 is indicative for highly purified virus (Westdijk et al.,2011). Finally, SDS-PAGE gels of the different serotypes show four majorprotein bands corresponding to the surface proteins of the poliovirus(FIG. 5). Hence, the purification process described herein is robust forthe purification of all three serotypes, irrespective of differences inthe surface properties of the serotypes and the virus titers at harvest.

TABLE 3 Quality of monovalent Inactivated Polio Virus and purifiedpoliovirus (SEC eluate) for the monovalent Inactivated Polio Virusmanufacturing process monovalent Inactivated Polio Virus SEC eluate D-HC- OD260/ Antigen TP/DU DNA OD280 Purity on SDS Serotypes (DU/mL)(μg/DU) (pg/DU) (—) page gel Mahoney 2014 0.008 <0.2 1.67 VP1, VP2, VP3and MEF-1 343 0.037 <1.2 1.75 VP4 are the major Saukett 1201 0.012 <0.31.71 bands (see FIG. 5)

Process performance is evaluated based on clearance of host cellimpurities, DNA and HCP as well as step yields for the differentproduction stages. Results are depicted in Table 4.

TABLE 4 Process performance of the monovalent Inactivated Polio VirusManufacturing process Process stage D-antigen recovery [%] HCP removal[%] DNA log removal Type 1 Type 2 Type 3 Type 1 Type 2 Type 3 Type 1Type 2 Type 3 Harvest 86 77 101 40 51 42 >5.6 >5.6 >5.5 Purification 4239 56 >99.9 >99.9 >99.9 * * * Inactivation 89 77 82 NA NA NA NA NA NAOverall 32 23 46 >99.9 >99.9 >99.9 >5.6 >5.6 >5.5 * Removal could not bedetermined as feed was already below detection level

For all three serotypes, residual HC-DNA levels after the clarificationstep are below the limit of quantification. Table 5 shows overallproductivity of poliovirus expressed in equivalent dose/ml cell culture.This calculation is based on a 40:8:32 ratio D-antigen Units/dose forpoliovirus types 1-3. Comparison of the final productivity afterinactivation shows that this process exceeds the current worldwide IPVmanufacturing VERO cell process platform, which yields 0.64, 1.04 and0.34 dose/ml virus culture for types 1-3, respectively (Kreeftenberg,2007). Hence, it can be concluded that despite the high initial impuritylevel in the feed originating from the high cell density harvest, a highresolution and high recovery process was developed, yielding unsurpassedhigh productivities for monovalent inactivated polio virus bulkproduction as an integral part of IPV vaccine manufacturing.

TABLE 5 Productivity of the 20 L monovalent Inactivated Polio VirusManufacturing process (# equivalent doses/ml cell culture) Type 1 Type 2Type 3 Product intermediate (Mahoney) (MEF-1) (Saukett) Crude harvest 6937 29 Clarified harvest 53 32 29 Purified harvest 22 12 16 Inactivatedpolio virus bulk 20 9.4 13

Example 6 Increased Poliovirus Purification Yields from Crude CellCulture Harvest by Addition of Different Cationic Detergents

PER.C6® cells were grown in a serum-free culture medium in a 10 Lbioreactor operated in perfusion mode to a cell density of approximately50×10⁶ vc/ml. Prior to infection with poliovirus type 2 (MEF-1), theculture was diluted with fresh culture medium to a viable cell densityof about 12.5×10⁶ vc/mL. The batch infection process took place in 10 Lbioreactors at 35° C., at a multiplicity of infection of 1. At the timeof harvest, 20-24 hours post-infection, a 120 ml sample was taken, whichwas subsequently distributed in 18 aliquots of 5 mL.

In order to determine the effect of a detergent on the polioviruscontaining crude cell harvests, a titration experiment was performedwith several cationic detergents; Hexadecyltrimethylammonium bromide(CTAB), Hexadecylpyridinium chloride (CPC) and Benzethonium chloride(BTC). A fixed amount of CTAB, CPC and BTC stock solutions (69, 70, 56mM, respectively, all including 40 mM NaCl) were added to the harvestaliquots at a targeted detergent concentration (between 0 and 4 mM). Thesamples were thoroughly mixed and incubated for one hour at 35° C.Subsequently, the samples were centrifuged for 5 minutes at 3000 g tospin down precipitated DNA. Supernatant samples were analyzed for virusquantity by D-antigen ELISA and for host cell DNA using Q-PCR.

FIG. 6 (Panel A) shows D-antigen release from poliovirus-containingcrude cell culture harvests as a result of the treatment with differentcationic detergents; CTAB, CPC and BTC, respectively. The D-antigenconcentrations in the supernatant, which are corrected for the detergentaddition dilution, are disclosed as a function of the detergentconcentration. FIG. 6 (Panel A) discloses that after the addition of adetergent (CTAB, CPC and BTC), the virus titer increased substantiallyas compared to before the addition of a detergent (CTAB, CPC and BTC).For each cationic detergent, the same pattern can be observed, i.e.,increasing the detergent (CTAB, CPC and BTC) concentration leads toincreased virus release from the crude cell harvest into the liquidphase.

FIG. 6 (Panel B) shows host cell DNA precipitation inpoliovirus-containing crude cell culture harvests as a result of thetreatment with a detergent (CTAB, CPC and BTC). The concentrations onthe y-axis have been corrected for the detergent dilution factor. Foreach cationic detergent, the same pattern can be observed, i.e., hostcell DNA is precipitated from the crude cell culture harvest. FIG. 6(Panel B) clearly indicates that effective DNA clearance occurred in thealiquots for detergent (CTAB, CPC or BTC) concentrations above 0.5 mM.

Since the increase of detergent did not lead to poliovirusprecipitation, a person skilled in the art would extrapolate theseresults to poliovirus-containing cell suspensions of even higher celldensities, e.g., of about 70×10⁶ cells/mL, e.g., of about 90×10⁶cells/mL, e.g., up to about 120×10⁶ cells/mL, e.g., up to about 150×10⁶cells/mL. The skilled person would conclude that the poliovirus fromsuch high cell density crude cell culture harvests can be purified bythe methods of this disclosure.

Example 7 Increased Poliovirus Purification Yields from Crude CellCulture Harvest by Addition of Different Types of Detergents (Anionic,Zwitterionic and Non-ionic)

PER.C6® cells were grown in a serum-free culture medium in a 10 Lbioreactor operated in perfusion mode to a cell density of approximately50×10⁶ vc/ml. Prior to infection with poliovirus, type 2 (MEF-1), theculture was diluted with fresh culture medium to a viable cell densityof about 12.5×10⁶ vc/mL. The batch infection process took place in 10 Lbioreactors at 35° C., at a multiplicity of infection of 1. At the timeof harvest, 20-24 hours post-infection, a 240 ml sample was taken, whichwas subsequently distributed in 42 aliquots of 5 mL.

In order to determine the effect of a detergent on thepoliovirus-containing crude cell harvests, a titration experiment wasperformed with several different types of detergents. Anionic detergents(Sodium taurodeoxycholate hydrate (STH) and Sodium dodecyl sulfate(SDS)), Zwitterionic detergents (3-(N,N-Dimethylmyristylammonio)propanesulfonate (SB3-14), and 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)), and Non-ionic detergents(4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON® X-100)and Decyl-β-D-1-thiomaltopyranoside (DTP)) were used as exemplarydetergents for their detergent class. A fixed amount of detergent stocksolutions were added to the harvest aliquots at a targeted detergentconcentration. The targeted detergent concentration for anionicdetergents (STH and SDS), zwitterionic detergents (SB3-14 and CHAPS),and for non-ionic detergents (TRITON® X-100 and DTP) was between 0 and 4mM. The samples of all detergent types (anionic, zwitterionic,non-ionic) were thoroughly mixed and incubated for one hour at 35° C.Subsequently, the samples were centrifuged for 5 minutes at 3000 g tospin down precipitated DNA. Supernatant samples were analyzed for virusquantity by D-antigen ELISA and for host cell DNA using Q-PCR.

FIG. 7 (Panels A, B, and C) shows D-antigen release frompoliovirus-containing crude cell culture harvests as a result of thetreatment with different types of detergents, anionic detergents (STHand SDS), zwitterionic detergents (SB3-14 and CHAPS) and non-ionicdetergents (TRITON® X-100 and DTP), respectively. The D-antigenconcentrations in the supernatant, which are corrected for the detergentaddition dilution, are disclosed as a function of the detergentconcentration. FIG. 7 (Panels A, B and C) discloses that after theaddition of a detergent (STH, SDS, SB3-14, CHAPS, TRITON® X-100 andDTP), the virus titer increased substantially as compared to before theaddition of a detergent (STH, SDS, SB3-14, CHAPS, TRITON® X-100 andDTP). For each type of detergent (anionic, zwitterionic or non-ionic),the same pattern can be observed, i.e., increasing the detergent (STH,SDS, SB3-14, CHAPS, TRITON® X-100 and DTP) concentration leads toincreased virus release from the crude cell harvest into the liquidphase. FIG. 8 (Panels A, B and C) shows host cell DNA release frompoliovirus-containing crude cell culture harvests as a result of thetreatment with a detergent (STH, SDS, SB3-14, CHAPS, TRITON® X-100 andDTP). The concentrations on the y-axis have been corrected for thedetergent dilution factor. For each type of detergent (anionic,zwitterionic or non-ionic), the same pattern can be observed, i.e.,increasing the detergent (STH, SDS, SB3-14, CHAPS, TRITON® X-100 andDTP) concentration leads to increased host cell DNA release from thecrude cell harvest into the liquid phase.

Since increase of concentration of the detergent types (anionic,zwitterionic or non-ionic) did not lead to poliovirus precipitation, aperson skilled in the art can extrapolate these results topoliovirus-containing cell suspensions of even higher cell densities,e.g., of about 70×10⁶ cells/mL, e.g., of about 90×10⁶ cells/mL, e.g., upto about 120×10⁶ cells/mL, e.g., up to about 150×10⁶ cells/mL. Theskilled person would conclude that the poliovirus from such high celldensity crude cell culture harvests can be purified by the methods ofthis disclosure.

Example 8 DB Treatment and Clarification as Part of the Sabin IPVPurification Train

This example describes the application of the harvest process (DBtreatment followed by cell clarification) as part of the purificationprocess of attenuated poliovirus serotypes (Sabin type 1, Sabin type 2and Sabin type 3) from crude cell culture harvests.

Cells, from the PER.C6® cell line, cells were grown in a serum-freeculture medium in a 10 L bioreactor operated in perfusion mode to a celldensity of approximately 50×10⁶ vc/ml. Prior to infection withpoliovirus serotype 1 (Sabin type 1), type 2 (Sabin type 2) or type 3(Sabin type 3), the culture was diluted with fresh culture medium to aviable cell density of 12.5×10⁶ vc/ml or 25×10⁶ vc/ml. Multiplicity ofinfection of 1 and 0.1 were used for the 12.5×10⁶ vc/ml and 25×10⁶ vc/mlcell cultures, respectively. In both cases, the batch infection processtook place in 10 L bioreactors at 32.5° C.

At the time of harvest (48 hours post-infection for Sabin type 1 orSabin type 3, and 72 hours post-infection for Sabin type 2), DB stocksolution was added to the bioreactors over a period of 30 minutes, to afinal DB concentration of 2.2 mM DB. After detergent addition, theDB-treated harvest (˜11 L) was mixed for 60 minutes. Finally, theDB-treated harvest was clarified and purified similarly as described forSalk IPV in FIG. 4 and Example 5.

Table 6 shows the overall D-Antigen recovery and HC-DNA removal of theDB treatment step followed by serial filtration. Table 7 summarizes thequality attributes of purified Sabin poliovirus.

TABLE 6 D-antigen recovery and HC-DNA concentration after DB treatmentand cell clarification step. VCDAI D-antigen HC-DNA Serotypes (×10⁶vc/ml) recovery (%) (ng/ml) Sabin type 2 12.5 126 <0.4 Sabin type 3 12.5105 <0.4 Sabin type 1 25 83 <0.4 Sabin type 2 25 76 <0.4 Sabin type 3 2585 <0.4

TABLE 7 Quality of purified Sabin polio virus before inactivation VCDAITP/DU HC-DNA OD260/OD280 Serotypes (×10⁶ vc/ml) (μg/DU) (pg/DU) (—)Sabin type 2 12.5 0.040 <1.3 1.72 Sabin type 3 12.5 0.004 <0.2 1.63Sabin type 1 25 0.009 <0.3 1.74 Sabin type 2 25 0.03  <1.1 1.67 Sabintype 3 25 —* <0.3 1.84 *Not available due to one or more missing data.

The results for the Sabin polio virus process show large similarity withthe results achieved for the wild-type strains. Also, for Sabin poliovirus strains, the combined DB treatment and clarification harvestprocess achieves high virus recovery with complete removal of HC-DNA(Table 6). Table 7 shows that the PER.C6®-based Sabin polio virus cellculture harvests could be sufficiently purified using the harvest andpurification process described in the disclosure. Residual specificprotein and DNA concentration meet regulatory requirements (WHO/EP). Inaddition, the absorbance ratio OD260/0D280 is indicative for highlypurified virus (Westdijk et al., 2011). Overall purity is the same aspurity obtained for wild-type polio virus strains (see Table 3 inExample 5).

The results are very promising, especially when one considers that thetwo types of viruses, wild-type and Sabin strains, differ in net surfacecharge (Thomassen et al., 2013). This once more demonstrates therobustness of the developed generic high productivity polio virusvaccine manufacturing process.

REFERENCES

Bakker W. A. M., Y. E. Thomassen, A. G. van't Oever, J. Westdijk, M. G.C. T. van Oijen, L. C. Sundermann, P. van't Veld, E. Sleeman, F. W. vanNimwegen, A. Hamidi, G. F. A. Kersten, N. van den Heuvel, J. T.Hendriks, and L. A. van der Pol. Inactivated polio vaccine developmentfor technology transfer using attenuated Sabin poliovirus strains toshift from Salk-IPV to Sabin-IPV. Vaccine 2011; 29(41):7188-96.

Cortin V., J. Thibault, D. Jacob, and A. Gamier. High-Titer PoliovirusVector Production in 293 S Cell Perfusion Culture. Biotechnol. Prog.2004.

European Pharmacopoeia 7.0, Poliomyelitis vaccine (inactivated).04/2010:0214.

Fuchs F., P. Minor, A. Daas, and C. Milne. Establishment of EuropeanPharmacopoeia BRP batch 2 for inactivated poliomyelitis vaccine for invitro D-antigen assay. Pharmeuropa Bio. 2003-1, 23-50, 2003.

Goerke A., B. To, A. Lee, S. Sagar, and K. Konz. Development of a NovelPoliovirus Purification Process Utilizing Selective Precipitation ofCellular DNA. Biotechnology and Bioengineering, Vol. 91, No. 1, Jul. 5,2005.

Henderson M., C. Wallis, and J. Melnick. Concentration and purificationof enteroviruses by membrane chromatography. Applied and EnvironmentalMicrobiology, November 1976, p. 689-693.

Kreeftenberg H., T. van der Velden, G. Kersten, N. van der Heuvel, andM. de Bruijn. Technology transfer of Sabin-IPV to new developing countrymarkets. Biologicals 2006; 34(2): 155-8.

Sanders B. A., D. Edo-Matas, J. H. H. V. Custers, M. H. Koldijk, V.Klaren, M. Turk, A. Luitjens, W. A. M. Bakker, F. UytdeHaag, J.Goudsmit, J. A. Lewis, and H. Schuitemaker, PER.C6® cells as aserum-free suspension cell platform for the production of high titerpoliovirus: a potential low cost of goods option for world supply ofinactivated poliovirus vaccine. Vaccine 2013; 31(5):850-6.

Thomassen Y. E., A. G. van't Oever, M. Vinke, A. Spiekstra, R. H.Wijffels, L. A. van der Pol, and W. A. M. Bakker. Scale down of theinactivated polio production process. Biotechnology and Bioengineering,Vol. 110 (5):1354-1365, May 2013.

Thomassen Y. E., G. van Eikenhorst, L.A. van der Pol, and W. A. M.Bakker. Isoelectric focusing with whole column imaging detection. Anal.Chem. 85:6089-6094 (2013).

D. H. W. Vlecken, R. P. M. Pelgrim, S. Ruminsk, W. A. M. Bakker, and L.A. van der Pol. Comparison of initial feasibility of host cell lines forviral vaccine production. Journal of Virological Methods 2013 193(2):278-283.

Westdijk J., D. Brugmans, J. Martin, A. van't Oever, W. A. M. Bakker, L.Levels, and G. Kersten. Characterization and standardization ofSabin-based inactivated polio vaccine: Proposal for a new antigen unitfor inactivated polio vaccines. Vaccine 2011; 29:3390-3397.

WHO technical report Series, No. 910, 2002. Recommendations for theproduction and control of poliomyelitis vaccine (inactivated).

Yuk I. H. Y., M. M. Olsen, S. Geyer, and S. P. Forestell. PerfusionCultures of Human Tumor Cells: A Scalable Production Platform forOncolytic Adenoviral Vectors. Biotechnol. Bioengin. 86:637-641 (2004).

The invention claimed is:
 1. A method of purifying poliovirus from acrude cell culture harvest containing poliovirus, said method comprisingthe steps of: a) adding a cationic detergent to the crude cell cultureharvest to obtain a mixture; and b) clarifying a mixture obtained fromstep a) to obtain a clarified harvest with poliovirus particles; whereinthe combination of step a) and step b) results in a reduction in hostcell DNA of at least 4 log
 10. 2. A method of enhancing poliovirusrelease from a crude cell culture harvest containing poliovirus, saidmethod comprising the steps of: a) adding a cationic detergent to thecrude cell culture harvest to obtain a mixture; and b) clarifying amixture obtained from step a) to obtain a clarified harvest withpoliovirus particles; wherein the combination of step a) and step b)results in a reduction in host cell DNA of at least 4 log
 10. 3. Amethod of purifying poliovirus from a crude cell culture harvestcontaining poliovirus, said method comprising the steps of: a) adding acationic detergent to the crude cell culture harvest to obtain amixture; b) clarifying a mixture obtained from step a) to obtain aclarified harvest with poliovirus particles; and c) subjecting theclarified harvest obtained in step b) to a capture step to obtain apoliovirus-containing suspension; wherein the combination of step a) andstep b) results in a reduction in host cell DNA of at least 4 log
 10. 4.The method of purifying poliovirus from a crude cell culture harvestaccording to claim 3, wherein said capture step is a cationic exchangechromatography step.
 5. The method according to claim 3, wherein thepoliovirus obtained in step c) is further separated from thepoliovirus-containing suspension by size exclusion.
 6. The methodaccording to claim 5, wherein said size exclusion is performed by sizeexclusion chromatography.
 7. A method of purifying poliovirus from acrude cell culture harvest containing poliovirus, said method comprisingthe steps of: a) adding a cationic detergent to the crude cell cultureharvest to obtain a mixture; b) clarifying a mixture obtained from stepa) to obtain a clarified harvest with poliovirus particles; c)subjecting the clarified harvest obtained in step b) to a cationicexchange chromatography step to obtain a poliovirus-containingsuspension; and d) further separating the poliovirus from thepoliovirus-containing suspension by size exclusion chromatography;wherein the combination of step a) and step b) results in a reduction inhost cell DNA of at least 4 log
 10. 8. The method according to claim 1,wherein said cationic detergent is selected from the group consisting ofHexadecyltrimethylammonium bromide (CTAB), Hexadecylpyridinium chloride(CPC), Benzethonium chloride (BTC) and domiphen bromide (DB).
 9. Themethod according to claim 8, wherein the cationic detergent is domiphenbromide (DB).
 10. The method according to claim 4, wherein thepoliovirus obtained in step c) is further separated from thepoliovirus-containing suspension by size exclusion.
 11. The methodaccording to claim 10, wherein the size exclusion is performed by sizeexclusion chromatography.